Uncontrolled Gli transcription factor activity causes several human cancers, including basal cell carcinoma, medulloblastoma, meningioma, and rhabdomyosarcoma. Oncogenic Gli function frequently stems from Hedgehog (Hh) pathway dysregulation, and chemical inhibitors of this developmental signaling pathway are now in clinical use. Despite these significant advances in our understanding and treatment of Gli- dependent cancers, current Hh pathway-targeting therapies have several limitations. First, nearly all Hh pathway-targeting drugs inhibit Smoothened (Smo), a G protein-coupled receptor-like protein that is required for Hh signal transduction. As a result, they are primarily effective against cancers caused by loss of Patched1 (Ptch1), a transmembrane repressor of Smo, or by certain activating mutations in Smo. Gli-dependent tumors initiated by downstream or parallel signaling mechanisms are insensitive to these compounds. Second, tumor regressions induced by Smo antagonists are often transient, as drug-resistant cancer cells can rapidly emerge. Third, Smo-targeting drugs can disrupt normal Hh pathway-dependent physiology, and preclinical studies further suggest that Smo blockade could cause developmental defects in children. Hh pathway inhibitors that act downstream of Smo and more directly suppress Gli function could overcome these constraints. In particular, compounds that selectivity inhibit Gli1 could be more general and effective anti-cancer agents, since this Gli isoform is a potent oncogene but dispensable for mammalian development and physiology. Toward this goal, our laboratory recently surveyed 325,120 compounds for their ability to inhibit the constitutive Gli activity in cells lacking Suppressor of Fused (Sufu), a direct Gli antagonist. Through this large-scale chemical screen, we have identified an imidazole derivative (glimidazole) that can inhibit Gli1 function but has no apparent effect on Gli2 or Gli3. We have also developed several glimidazole analogs with nanomolar potencies in cell-based assays, used photoaffinity labeling to discover a specific glimidazole-binding protein that may link mitochondrial signaling and Gli1 regulation, and demonstrated the ability of these Gli1-selective inhibitors to block tumor growth in vitro and in vivo. We are now pursuing the next steps required to establish glimidazole-based compounds as a new class of Hh pathway-targeting chemotherapies. Our aims are: (1) to characterize the glimidazole target discovered in our photoaffinity labeling experiments and determine its roles in Gli1 regulation; (2) to develop glimidazole analogs with optimized potency, target selectivity, and pharmacokinetic properties; and (3) to compare the efficacy of selected glimidazole-class molecules to Smo antagonists in murine models of medulloblastoma. Taken together, our studies will provide new insights into the mechanisms of Gli1 regulation, uncover novel targets for anti-cancer therapies, and yield chemical leads for future drug development efforts.